Title page i - DORAS - Dublin City University

Document Sample
Title page i - DORAS - Dublin City University Powered By Docstoc
					     Chemical and Degradation
Characteristics of Soil Microbial

Adrian Spence M.PhiL., B.Sc. (First Class Honours),
                 A.Sc., AMRSC

 A thesis presented for the degree of Doctor of Philosophy

                   Dublin City University

             Ollscoil Chathair Bhaile Átha Cliath
                 School of Chemical Sciences

                         June 2010


I hereby certify that this material, which I now submit for assessment on the programme
of study leading to the award of PhD is entirely my own work, that I have exercised
reasonable care to ensure that the work is original, and does not to the best of my
knowledge breach any law of copyright, and has not been taken from the work of others
save and to the extent that such work has been cited and acknowledged within the text
of my work.

Signed:       __________________
              Adrian Spence

ID No.:       56114397

Date:         _________________


        I would like to thank my supervisor, Dr. Brian P. Kelleher for the wonderful
opportunity to conduct research under his supervision, for his leadership by example, his
support and encouragement at both an academic level and a personal level.

I would also like to express my profound gratitude to:
        The Environmental Protection Agency (EPA) of Ireland, Science Foundation of
Ireland (SFI) and the Geological Survey of Ireland (GSI) for funding this research;

        The Teagasc Agricultural Research Station, Carlow, Ireland for providing soil
samples for experimental work;

        Dr. Andre J. Simpson in the Department of Physical and Environmental
Sciences, University of Toronto at Scarborough, Canada for conducting high resolution
NMR analysis;

        Dr. Michael Oelgemoeller, formerly of the School of Chemical Sciences for the
use of his UV-radiation chamber to conduct UV-experiments;

        The Richard O’kennedy Research Group in the School of Biotechnology for
allowing me the use of their PCR machine;

        Mr. Barry O’Connell in the National Centre for Sensory Research for X-ray
diffraction training;

        The entire technical staff of the School of Chemical Sciences, especially,
Damien, John, Ambrose, Veronica, and Mary;

        Special thanks to my fellow postgraduate and postdoctoral colleagues for their
invaluable contributions;

        Finally, but by no means least, to my wife Samantha for her unwavering support
over the years.

       Soil organic matter (SOM) contains substantial amounts of carbon and nitrogen and
plays an important role in regulating anthropogenic changes to the global carbon and
nitrogen biogeochemical cycles. It also plays essential roles in agricultural productivity,
water quality, immobilization and transport of nutrients and anthropogenic chemicals, while
also concealing exciting opportunities for the discovery of novel compounds for potential
use in industry and medicine. Despite these critical roles and potential, many uncertainties
exist regarding the size of the labile and refractory SOM pool, carbon dynamics within the
SOM pool, and the role of SOM in carbon sequestration. Several studies have investigated
the contribution of plant litter and crop residues to SOM. However, the contribution of soil
microbial biomass to carbon-cycling is seriously underestimated and its turnover poorly

       To address these knowledge gaps, a study utilizing 13C and 15N isotopically enriched
soil microbial biomass was undertaken to determine the transformation dynamics of major
microbial (bio)macromolecules degraded under ambient and prolonged ultraviolet (UV)
conditions. The application of advanced nuclear magnetic resonance (NMR) approaches
revealed a relative increase in aliphatic-C with a concomitant decrease in carbohydrates and
proteinaceous materials, indicating that aliphatic species are selectively preserved. Further
analysis of the lipid fractions by gas chromatography-mass spectrometry (GC-MS) indicated
that n-alkanes, mono- and poly-unsaturated n-C16 and n-C18, and fatty acids having chains of
less than 14 C are relatively labile. Conversely, saturated n-C16 and n-C18 are highly
recalcitrant. Bio-refractory proteins surviving degradation were subsequently identified as
membrane proteins and enzymes using in-gel tryptic digestion, matrix assisted laser
desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and/or
electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) analysis. To
determine the role of soil minerals in SOM turnover, clay-microbial complexes were
investigated. X-ray diffraction analysis coupled with scanning electron microscopy,
elemental X-ray analysis and NMR spectroscopy revealed that aliphatic species, proteins
and carbohydrates are preferentially adsorbed to clay surfaces where they are physically
protected from degradation.

Table of contents:
      Title page                                                            i
      Declaration                                                           ii
      Acknowledgements                                                      iii
      Abstract                                                              iv

Chapter 1 Introduction and literature review
1.1   Introduction                                                          2
      1.1.1   Significance of this topic                                    2
      1.1.2   Understanding of the issues and their impacts on the global   4
      1.1.3   Microbes and microbial degradation                            5
      1.1.4   Soil organic matter                                           7
      1.1.5   Dissolved organic matter                                      9
      1.1.6   Lipids as a source of soil organic carbon                     11
      1.1.7   Soil organic nitrogen                                         13
      1.1.8   Stable carbon isotope biogeochemistry                         18
      1.1.9   Phosphorus and the global biogeochemical cycling of carbon    20
      1.1.10 Silicon and the global carbon cycle                            22
      1.1.11 Ultraviolet induced degradation of soil organic matter         23
      1.1.12 Photodegradation of microbial components                       25
      1.1.13 Clay-organo interactions                                       27
      1.1.14 Mechanism of clay-organic matter interactions                  28
      1.1.15 Mechanism of organic matter protection                         29
      1.1.16 Clay-microbial interactions                                    32
      1.1.17 Clay minerals                                                  33
      1.1.18 Project objectives                                             34
1.2   References                                                            36

Chapter 2 Microbial characterization
2.1   Introduction                                                          57
2.2   Materials and Method
      2.2.1   Soil and sampling                                             58
      2.2.2   Media and growth conditions                                   58
      2.2.3   Total DNA isolation and PCR amplification                       59
      2.2.4   Nucleotide sequence and phylogenetic analysis                   60
2.3 Results and Discussion
      2.3.1   PCR amplification                                               61
      2.3.2   Nucleotide sequence and phylogenetic analysis                   61
2.4   References                                                              70

Chapter 3 Assessing the fate and transformation of microbial
residues in soil
3.1   Introduction                                                            74
3.2   Materials and Methods
      3.2.1   Media and growth conditions                                     75
      3.2.2   Decomposition experiment                                        76
      3.2.3   High resolution magic angle spinning (HR-MAS) NMR               77
      3.2.4   Phosphorus extraction                                           79
      3.2.5   Solution 31P NMR spectroscopy                                   79
3.3   Results and Discussion
      3.3.1       C NMR analysis of microbial biomass                         80
      3.3.2       H and Diffusion Edited NMR analysis of microbial biomass    82
      3.3.3       C HSQC NMR analysis of microbial biomass                    84
      3.3.4   Quantitative analysis                                           86
      3.3.5   Nitrogen NMR analysis of microbial biomass                      90
      3.3.6       C NMR analysis of microbial leachate                        92
      3.3.7       C HSQC NMR analysis of microbial leachate                   95
      3.3.8       H and Diffusion Edited NMR analysis of microbial leachate   97
      3.3.9   Nitrogen NMR analysis of microbial leachate                     102
      3.3.10 Solution P NMR analysis of microbial biomass                     104
3.4   Conclusions                                                             113
3.5   References                                                              115

Chapter 4 Clay-organo interactions
4.1   Introduction                                                            125
4.2   Materials and Methods
      4.2.1   Microbial propagation and bacterial growth on clay              126

      4.2.2   Protein extraction                                             126
      4.2.3   Determination of protein concentration                         127
      4.2.4   Sorption experiments                                           127
      4.2.5   Direct microscopy and elemental analysis                       128
      4.2.6   X-ray diffraction analysis                                     128
      4.2.7   Acid hydrolysis                                                128
      4.2.8   High resolution magic angle spinning (HR-MAS) NMR              129
4.3   Results and Discussion
      4.3.1   Equilibrium adsorption                                         129
      4.3.2   Scanning electron microscopy and elemental analysis            130
      4.3.3   X-ray diffraction analysis                                     132
      4.3.4   NMR analysis of clay-organo complexes                          134
      4.3.5   NMR analysis of acid hydrolyzed clay-microbial complexes       140
      4.3.6   NMR analysis of microbial protein and clay-protein complexes   142
4.4   Conclusions                                                            144
4.5   References                                                             146

Chapter 5 Degradation of microbial lipids and amino acids
5.1   Introduction                                                           153
5.2   Materials and Methods
      5.2.1   Microbial propagation and bacterial growth on clay             154
      5.2.2   Decomposition experiment                                       154
      5.2.3   Solvent extraction                                             154
      5.2.4   Acid hydrolysis (AHY)                                          155
      5.2.5   Derivatization                                                 155
      5.2.6   GC-MS analysis                                                 155
5.3   Results
      5.3.1   Lipid analysis of ambient degraded microbial biomass           156
      5.3.2   Lipid analysis of UV degraded microbial biomass                161
      5.3.3   Lipid analysis of ambient degraded microbial leachates         163
      5.3.4   Lipid analysis of UV degraded microbial leachates              166
      5.3.5   Amino acid analysis of ambient degraded microbial biomass      169
      5.3.6   Amino acid analysis of UV degraded microbial biomass           172
      5.3.7   Lipid analysis of ambient degraded montmorillonite-complexes   174

      5.3.8   Lipid analysis of UV degraded montmorillonite-complexes        178
      5.3.9   Lipid analysis of ambient degraded montmorillonite-microbial   180
      5.3.10 Lipid analysis of UV degraded montmorillonite-microbial         183
      5.3.11 Amino acid analysis of ambient degraded montmorillonite-        184
      5.3.12 Amino acid analysis of UV degraded montmorillonite-complexes    187
5.4   Discussion
      5.4.1   Lipid analysis                                                 188
      5.4.2   Photooxidation and autoxidation of microbial lipids            191
      5.4.3   Degradation of polysaccharides and diterpenes                  195
      5.4.4   Degradation of sterols                                         196
      5.4.5   Analysis of acid hydrolysable compounds                        197
5.5   Conclusions                                                            199
5.5   References                                                             201

Chapter 6 Protein degradation and peptide mass fingerprinting
6.1   Introduction                                                           208
6.2   Materials and Methods
      6.2.1   Protein extraction                                             210
      6.2.2   Protein separation                                             210
      6.2.3   In-gel protein digestion                                       210
      6.2.4   MALDI-Q-ToF MS analysis                                        211
      6.2.5   Database interrogation                                         211
      6.2.6   Molecular weight analysis by LC-ESI-TOF-MS                     212
6.3   Results and Discussion
      6.3.1   Analysis of proteins from microbial biomass and microbial      213
      6.3.2   Analysis of proteins isolated from clay-microbial complexes    215
      6.3.3   Validation of peptide mass fingerprinting                      220
      6.3.4   Limitations of peptide mass fingerprinting                     221
      6.3.5   Peptide mass fingerprinting of refractory proteins             222
6.4   Conclusions                                                            230

6.5    References                    232

Chapter 7 General Conclusions
7.1    General conclusions           238
7.2    References                    245

List of abbreviations                247


Shared By: